Mutation of Phosphorylation Sites S1024 and T1026

After performing the first step of double mutagenesis PCR, the products I and II were run on an agarose gel and cut out. In the second step, a PCR reaction was set up containing the purified fragments. For obtaining a higher yield of the desired DNA fragment a third PCR step was done. This reaction was also run on a gel, cut out and Gene-cleaned. Following digestion of both the plasmid and the fragment, the plasmid was treated with calf intestine phosphatase to prevent a recircularization. The enzyme

removes the 3’-phosphate group so that the ligase is unable to connect both ends of the plasmid. In order to remove this enzyme from the subsequent ligation step, a Gene-clean procedure without the TBE-modifier was performed.

For successful ligation it is necessary to mix the cut plasmid and the cut fragment at approximately 1:3 ratio. Therefore, I ran an aliquot of the plasmid and the fragment on a 1.5% agarose gel to estimate the concentration of each. The ligation reaction (10 µl) was then set up for at least 4 hours at room temperature.

Then DH10β E.coli cells were transformed with the ligation mix and spread onto a L- agar plate containing kanamycin at 30 µg/ml. Plates were incubated at 37°C overnight.

Colonies resulting from the transformation were screened by PCR with T7- promoter and T7- terminator primers. Positive clones, detected by agarose gel electrophoresis, were used to inoculate 5 ml LB medium containing kanamycin at 30 µg/ml, which was grown at 37°C overnight with shaking. One ml was used to inoculate 400 ml LB medium containing kanamycin at 30 µg/ml, which were incubated overnight on a shaking platform at 37°C.

The harvested pellet was used to purify the desired plasmid with the QIAprep Spin Maxiprep Kit (Quiagen UK Ltd).

The sequence of the plasmid was compared using Megalign^TM (DNASTAR Inc.) to the published sequence to ensure both that the engineered mutations were present and that no errors have been incorporated during the PCR mutagenesis procedure.

The verified plasmid was used to transform BL21(DE3)pLysS cells and these were plated on L-agar plates containing appropriate antibiotics (chloramphenicol 25 µg/ml;

kanamycin 30 µg/ml) and incubated overnight at 37°C.

One colony was used to inoculate 100 ml LB medium containing appropriate antibiotics, which were incubated at 37°C on a shaking platform overnight. From that culture 15 ml were used to inoculate 800 ml LB medium containing appropriate antibiotics. After adding IPTG to a concentration of 0.4mM the protein was expressed. Cells were harvested usually after 3 hours protein expression and the pellet stored at -80°C.

The C8 mutant was purified using the same protocol as for the wild type protein.

Figure 3–5: purification mutant C8 (S1024D;T1026D) domain of cMyBP- C in pET28a under denaturing conditions, after Gel filtration column; fractions 4-9 pure

Now the mutant domain was dialyzed overnight in phosphorylation assay buffer, which did not contain urea. After assessing the quantity of protein and running a sample on a SDS-PAGE gel, the phosphorylation reaction was performed. It was carried out using γ ³²P-ATP and AMP-activated kinase (AMPK). Based on the prediction of Carling and co-workers, we expected a significant reduction of phosphorylation of the mutated domain compared with wild type. However, as shown in Figure 3–6 the mutant protein is still phosphorylated to a similar level as wild type.

This experiment disproves the hypothesis that either S1024 or T1026 serves as a substrate for AMPK in vitro.

Figure 3–6: Autoradiography of C8 WT and C8mt (S1024D;T1026D) still phosphorylated in a similar intensity as the WT domain; C8mt (S1024D;T1026D) is later labelled in this chapter mt F

Relying on the data that I got from the phosphorylation experiment of the C8 WT, the most logical step to do at this stage was performing a phosphoamino acid analysis to show whether it was serine and/or threonine residues that were modified. The basic principle of this assay is the two dimensional separation of the amino acids of a ³² P-labelled protein after acid hydrolysis.

Figure 3–7: Phosphoamino acid analysis C8 WT domain

pH 3.5

pH 1.9

This experiment showed conclusively that the only phosphorylated amino acid in the labelled C8 domain was serine. The next consequent step had to be the identification of the other serines and the mutation of these to unphosphorylatable alanine residues.

Two additional serines were identified: Ser1020 and Ser1040.

L P R H L R Q T I Q K K V G 975 CTG CCC AGG CAC CTG CGC CAG ACC ATT CAG AAG AAG GTC GGG

GAC GGG TCC GTG GAC GCG GTC TGG TAA GTC TTC TTC CAG CCC E P V N L L I F Q G K P R P 989 GAG CCT GTG AAC CTT CTC ATC CCT TTC CAG GGC AAG CCC CGG

CTC GGA CAC TTG GAA GAG TAG GGA AAG GTC CCG TTC GGG GCC Q V T W T K E G Q P L A G E 1003 CCT CAG GTG ACC TGG ACC AAA GAG GGG CAG CCC CTG GCA GGC

GGA GTC CAC TGG ACC TGG TTT CTC CCC GTC GGG GAC CGT CCG E V P S I R N S P T D T I L 1017 GAG GAG GTG AGC ATC CGC AAC AGC CCC ACA GAC ACC ATC CTG

CTC CTC CAC TCG TAG GCG TTG TCG GGG TGT CTG TGG TAG GAC F I R A A R R V H S G T Y Q 1031 TTC ATC CGG GCC GCT CGC CGC GTG CAT TCA GGC ACT TAC CAG

AAG TAG GCC CGG CGA GCG GCG CAC GTA AGT CCG TGA ATG GTC

V T V R I E N M E D K A T L 1045 GTG ACG GTG CGC ATT GAG AAC ATG GAG GAC AAG GCC ACG CTG

CAC TGC CAC GCG TAA CTC TTG TAC CTC CTG TTC CGG TGC GAC V L Q V V D K P

1059 GTG CTG CAG GTT GTT GAC AAG CCA CAC GAC GTC CAA CAA CTG TTC GGT

Figure 3–8: MyBP-C domain C8 sequence showing the serine residues highlighted in red.

3.2.4 Cloning, Expression and Purification of other C8 Mutant